A Comparative Study of Cathepsin D Expression in Peripheral and Central Giant Cell Granuloma of the Jaws by Immunohistochemistry Technique.

STATEMENT OF THE PROBLEM
Peripheral and central giant cell granuloma are two common benign lesions of the oral cavity. In spite of histopathological similarities, they have different clinical behaviors. Cathepsin D is a lysosomal enzyme which has different functions on the basis of protein and applied peptide cleavage.


PURPOSE
This research aimed to evaluate and compare the expression level of Cathepsin D in these two lesions to find the reasons for the differences in clinical and biologic characteristics.


MATERIALS AND METHOD
The expression of Cathepsin D was investigated by using the immunohistochemistry method in 20 samples of peripheral giant cell granuloma and 20 samples of central giant cell granuloma. The percentage of stained giant cells (labeling index), the intensity of staining of giant cells, and staining-intensity-distribution in both groups were calculated and compared.


RESULTS
The labeling indices of Cathepsin D in peripheral giant cell granuloma and central giant cell granuloma were 95.9±4.03 and 95.6±2.34, respectively. There was no significant difference in the percentages of stained giant cells between the two groups (p= 0.586). The intensity of staining of giant cells in central giant cell granuloma was stronger than that of peripheral giant cell granuloma (p> 0.001). Staining- intensity- distribution of giant cells in central giant cell granuloma was significantly greater than that of the peripheral type of lesion (p= 0.001).


CONCLUSION
The higher expression level of Cathepsin D in central giant cell granuloma compared to peripheral type of lesion can explain more aggressive behavior of central giant cell granuloma.


Introduction
Peripheral giant cell granuloma (PGCG) occurs as a red or purple nodule exclusively on the gingiva and alveolar ridge. These lesions originate from the periodontal ligament or mucoperiosteum of the alveolar ridge as a result of local irritation or trauma. [1] PGCG can develop at any age, especially during the fifth and sixth decades of life with a slight female predilection. [1][2] In some cases, PGCG affects the underlying bone and may cause cupping resorption. [2] Central giant cell granuloma (CGCG) occurs within the jaw bones and appears as radiolucent defects in radiographs which may be unilocular or multilocular. [3] The majority of these lesions are noted in young adults with a predilection for females. [4][5] Even some case of this lesion have been associated with significant asymmetries, [6][7] repeated recurrences, [8] multifocal incidences, [8][9] invasion and extensive destruction of jaw bones. [10] Both CGCG and PGCG exhibit similar histopathological features, and are characterized by the presence of abundant mononuclear stromal cells, admixed with a large number of multinucleated giant cells and a rich vascularized stroma with extravasated erythrocytes, hemosiderin deposition, and blood. Meanwhile, these lesions may have different clinical behaviors. [11][12] Although various parameters have been compared between these two lesions in different studies, the reasons behind the differences in the biologic behaviors of these lesions are still to be elucidated. [3,[13][14] Cathepsin D is a soluble lysosomal aspartic endopeptidase which is released from the rough endoplasmic reticulum as preprocathepsin D and after elimination of the signal peptide, procathepsin D is carried into the intracellular vesicular structures. The various physiologic functions of Cathepsin D depend on its capacity to cleave functional and structural proteins and peptides. [15] Research has shown the presence of Cathepsin D in the vacuoles and vesicles of alveolar bone osteoclasts in mice, indicating that they are necessary for bone resorption and remodeling. [16][17] In addition, it has been demonstrated that giant cells in the giant cell tumor of long bones contain Cathepsin D. This enzyme has a positive relationship with the local invasion of this tumor, which might be attributed to its role in bone resorption. [18] Given the presence of Cathepsin D in osteoclasts and its effect on resorption of bone, the present study was undertaken to evaluate and compare its incidence through an immunohistochemical technique in PGCG and CGCG in an attempt to explain the reasons for different biological behaviors of these two tumors.

Materials and Method
The present descriptive analytical study was carried out by using a cross-sectional design. A total of 40 samples

Immunohistochemical Technique
The indirect immunohistochemical technique to stain for Cathepsin D consisted of the following steps: 1. Deparaffinization step: the slides with the specific tissue were placed in an oven for 60 minutes and heated to 60° C. They were then immersed in xylol and descending concentrations of alcohol (90%, 80% and 70%, respectively) for the tissue water absorption in preparation for the test.

Immunohistochemical evaluation
The stained giant cell counts were determined by count- In addition, the Cathepsin D immunoreactivity was evaluated semi-quantitatively and scored based on the percentage of stained cells ; score 0 when <1% of cells were stained, and score 1 to 4 when 1-25%, 26-50%, 51-75%, and >75% of cells were stained respectively.
Staining intensity was evaluated and scored as 0 (no cell staining), 1 (weak staining), 2 (moderate staining), and 3 (intense staining) based on the stating strength of the cells. [19] The scores were determined in each field. In order to achieve more accurate results and more reliable determination of staining intensity in the whole slide, 8 fields were scored separately and their means were calculated.
Staining intensity distribution (SID) was calculated separately for each field by multiplying the percentage of giant cells stained in that field by the intensity of staining in that field; the mean SID was calculated for each sample.

Statistical Analyses
Statistical analyses were carried out by using SPSS software, version 16. Kolmogorov-Smirnov was used to determine the distribution of the three variables of stained cells, staining intensity, and SID.
T test was used to evaluate Cathepsin D LI and SID between the two groups. Fisher's exact test was used to evaluate and compare the staining intensity of giant cells between the two groups.

Results
By assessment of study group documents, the patients' information such as age, sex, and site of the lesions were extracted. (Tables 1 and 2)

Immunohistochemical findings
Positive immunostaining giant cells had cytoplasmic brown staining in the two groups (Figures 1 and 2).

Discussion
In the present study, expression of Cathepsin D was    Based on the available data and a literature review, this study appears to be the first in which Cathepsin D was evaluated in PGCG and CGCG quantitatively and semi-quantitatively. Hence, the mechanisms suggested in the present study to explain a higher rate of expression of Cathepsin D in CGCG compared to PGCG should be further evaluated and confirmed by further studies. In addition, given the complexities and the extent of involved cellular-molecular mechanisms, the effect of these currently-unknown factors cannot be ruled out.

Conclusion
The number of stained giant cells was similar in both groups (CGCG and PGCG) in the present study. The giant cells in CGCG stained more intensely than in PGCG. SID in the CGCG was significantly different from that in the PGCG group; it was higher in the former group. There was no relationship between the expression of Cathepsin D in the PGCG and CGCG giant cells and age and sex.